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One‐step, PCR‐mediated, gene disruption in the yeast Hansenula polymorpha
Author(s) -
González Celedonio,
Perdomo Germán,
Tejera Paula,
Brito Nélida,
Siverio José M.
Publication year - 1999
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(19990930)15:13<1323::aid-yea459>3.0.co;2-1
Subject(s) - ura3 , biology , gene , marker gene , genetics , locus (genetics) , saccharomyces cerevisiae , gene targeting , heterologous , microbiology and biotechnology
Previous evidence based on the experience of our laboratory showed that one‐step gene disruption in the yeast Hansenula polymorpha is not straightforward. A systematic study of several factors which could affect gene disruption frequency was carried out. We found that the more critical factor affecting one‐step gene disruption in H. polymorpha is the length of the target gene region flanking the marker gene. Target gene regions of about 1 kb flanking the marker gene were necessary to obtain a disruption frequency of about 50%. However, the gene marker, either homologous or heterologous, the locus and the strain examined did not significantly affect the frequency of disruption; the highest disruption frequency obtained for the YNR1 gene was in the strain HMI39, using the Saccharomyces cerevisiae URA3 gene as a marker. Since long regions flanking the gene marker do not allow the easy PCR‐mediated strategies, developed for S. cerevisiae , to obtain constructs to disrupt a given gene in H. polymorpha, an alternative PCR strategy was developed. Copyright © 1999 John Wiley & Sons, Ltd.